Protein or polypeptide, method for producing the same and intermediate compound therefor

ABSTRACT

The present invention provides a protein or polypeptide substance having at least one amino group bonded to a polyethyleneglycolosy group represented by the following general formula; 
     
         R.sub.1 --(OCH.sub.2 CH.sub.2).sub.n --O-- 
    
     (wherein R 1  represents an optionally substituted cholesteryl group; and n represents a positive integer which is arbitrarily variable), and a method for producing said the protein or polypeptide. 
     The present invention also provides a reactive polyethyleneglycol derivative as an intermediated for above the method. 
     The chemically modified protein or polypeptide of the present invention never impairs bonding with a receptor and has a high plysiological activity. Then, a behaviors in vivo of these substance is improved. Now medical substances or drugs having a high pharmacological activity can be developed by using these substance.

This is a national stage application of PCT/JP95/00968 filed May 19,1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel protein or polypeptide modifiedin bonding with a polyethylene glycol derivative, which is useful as aphysiologically active or medically active substance or an intermediatecompound therefor, and a method for producing the same. The presentinvention also relates a reactive polyethylene glycol derivative whichis an intermediate compound for the novel protein or polypeptideabove-mentioned.

2. Description of the Related Art

Many protein, polyaminoacid and peptide substances having aphysiological or medical activity have recently been discovered,permitting expectation of more common application to medical substance.However, these protein and peptide substances have only a shorthalf-life in blood when administered in vivo, giving a sufficientpharmacological effect in few cases. In order to utilize thesesubstances as medical substance, therefore, it is believed to beabsolutely necessary to improve behaviors in vivo by some method orother.

It is known that many of physiologically active or medically activesubstances administered in vivo, particularly into the blood flow,disappear from the biological body through glomerular filtration in thekidney. This glomerular filtration process may be considered a kind ofmolecular sieve in principle: substances of a molecular weight smallerthan that of albumin (about 60,000) which is plasma proteinindispensable for a biological body are excreted as a rule. In order toimprove in vivo behaviors of protein and peptide medical substancedisappearing from the body through glomerular filtration, therefore, ithas conventionally been believed to be necessary to increase themolecular weight of medical substance through various chemicalmodifications.

As a method for improving behaviors in vivo of a protein or peptidemedical substance, chemical modification using a water-soluble polymertypically represented by polyethylene glycol (hereinafter abbreviated as"PEG" as required) has popularly been applied. PEG has rather a longhistory: this substance has been utilized and studied widely in variousareas since its synthesis in 1859. In the areas of biochemistry andmedicines or medical drugs as well, it is confirmed that PEG exerts nointeraction to protein except for physical stereo-hindrances, and nochange is observed in protein CD spectrum even in a high-concentrationaqueous PEG solution. This suggests that modification with PEG does notdestroy the higher-order structure of protein. When, for example, PEG ofa molecular weight of 4000 is administered to a dog at a rate of 90mg/kg/day for a period of a year, no abnormality is observed in bodyweight or in a pathological or hemological inspection. Administration ofPEG to a guinea pig does not cause an allergic symptom. Safety of PEG isthus commonly confirmed. By modifying protein with PEG having the uniqueproperties as described above, it is expected to be possible to developa protein or peptide medical substance having a long life in vivo, whichis hard to be recognized not only by the immune system but also by thereticuloendothelial system.

When modifying a protein or peptide medical substance with PEG, thefollowing advantages are available. When non-denatured protein isinsoluble under a physiological pH condition or only partially soluble,it is possible to considerably improve solubility under thephysiological pH condition by modifying protein with PEG, and also toreduce immune response of non-denatured protein. For these advantages,many types of protein or peptide medical substance have been modifiedwith PEG to date for utilization in medical drugs regarding generalremarks, see Inada, Y., Yoshimoto, T., Matsushima, A. and Saito, Y.(1986) Trends Biotechno, 1, 4: 68-73!.

PEG derivatives so far used for modifying protein or peptide medicalsubstance include: 2-(alkoxypolyethyleneglycoxy)-4,6-dechlorotriadineAbuchowski, A., Van Es, T., Palczuk, N. C. and Davis, F. F. (1977) J.Biol. Chem. 252, 3578-3581!;6-(alkoxypolyethyleneglycoxy)-S-carboxamide-methyl-dithiocarbonate King,T. P. and Weiner, C. (1980) Int. J. Peptide Protein Res. 16, 147-155!;2-(alkoxypolyethylene-glycoxy)-N-succinimyzilsuccinate Abuchowski, A,Kazo, G. M., Verhoest, C., Van Es, T., Kafkewitz, D., Viau, A. andDavis, F. (1984) Cancer Biochem. Biophys. 7, 175-186!;2-(alkoxypolyethyleneglycoxy) carboxyimidazole Beauchamp, C. O., Gonias,S. L., Menapace, D. P. and Pizzo, S. V. (1983) Anal. Biochem. 131,25-33!; 2-alkoxypolyethyleneglycoxy)-2,4,5-trichlorobenzene Versonese,F. M., Largajolli, R., Boccu, E., Benassi, C. A. arid Schiavon, O.(1985) Applied Biochem. Biotech., 11, 141-152!;2-(alkoxypolyethylene-glycoxy)-4-nitrobenzene Versonese, F. M.,Largajolli, R., Boccu, E., Benassi, C. A. and Schiavon, O. (1985)Applied Biochem. Biotech. 11, 141-152!;2-(alokxypolyethylene-glycoxy)-2,2,2-trifluoroethane Delgads, C., Patel,J. N., Francis, G. B. and Fisher, D. (1990) Biotech. Applied Biochem.12, 119-128!; 2-(alkoxypolyethylenealdehyde) Andrews, B. A., Head, D.M., Dunthrone, P. and Asenjo, J. A. (1990) Biotech. Tech. 4, 49-54!; and2-alkoxypolyethylene-glycoxymethylepoxide Andrews, B. A., Head, D. M.,Dunthrone, P. and Adenjo, J. A. (1990) Biotech. Tech. 4, 49-54!.

Regarding modification with a PEG derivative, a number of patentapplications have been filed to date. Some examples include, forexample, Japanese Provisonal Patent Publication JP-A-61-178,926;Japanese Provisional Patent Publication JP-A-61-249,388; JapaneseProvisional Patent Publication JP-A-1-316,400; Japanese ProvisionalPatent Publication JP-A-2-117,920; Japanese Provisional PatentPublication JP-A-3-88,822; Japanese Proisional Patent PublicationJP-A-5-117,300; Japanese Provisional Patent Publication JP-A-132,431;Japanese Provisional Patent Publication JP-A-5-214,092; and JapaneseProvisional Patent Publication JP-A-5-503,092. All these inventions arebased on attention to the high water solubility of PEG, having thereforea terminal structure comprising a short-chain aliphatic group.

However, the method of improving behaviors in vivo by increasing themolecular weight of a protein or peptide medical substance through PEGmodification is considered to suffer on the other hand limitationsimposed on medical substances to which it is applicable. For example,most of conventional protein or peptide medical substances of which PEGmodification has been confirmed to have improved behaviors in vivo andincreased pharmacological effect are limited to enzymes. A conceivablecause is the pharmacological effect expressing mechanism unique toenzymes.

An enzyme having a physiological activity expresses its effect bycausing a substance detrimental for a biological body or a substancespecifically required at a site suffering from a disease such as tumorto specifically disappear through a chemical reaction such asmetabolism. In the case of PEG modification of an enzyme, therefore, itis possible to increase the pharmacological effect by only causingenzyme molecules to be present in blood for a long period of time.However, such a method of increasing the molecular weight of a medicalsubstance through PEG modification is not clearly a method commonlyapplicable to all protein or peptide medical substance other thanenzymes.

More particularly, a protein or peptide medical substances requiringimprovement of behaviors in vivo necessitates bonding with a receptor,for example, for the purpose of displaying its pharmacological effect,PEG modification cannot be expected to increase the pharmacologicaleffect, but the pharmacological effect decreases in many cases as aresult of impairment of receptor bonding caused by stress hindrance ofpolymer PEG used for modification. For example, in Ehrat, M, and Luisi,P. L. (1983) Biopolymer 22, 569, 573, when PEG-modifying insulins to bebonded with a receptor for more remarkable pharmacological effect, adecrease in pharmacological effect in vivo by up to 50% is reported ascompared with non-modified insulin.

Therefore, for many of protein, polyaminoacid and peptide substancesexpected to become applicable to medical uses, bonding with a receptoror the like is a necessary prerequisite for the expression ofpharmacological effect. Under such circumstances, there is an increasingdemand for achievement of a novel method of improving behaviors in vivoof medical substances, which does not impair bonding with a site where apharmacological effect is to be expressed, such as a receptor or thelike, i.e., novel means for chemical modification, and a medicalsubstance comprising a pharmacologically active substance modified bysuch means.

SUMMARY OF THE INVENTION

The present invention has therefore an object to provide a novelchemically-modified physiologically active or medically active substancewhich never impairs bonding with a receptor or the like, a medicalsubstance comprising the same, and novel means of modification for suchsubstance and medical drug.

The present invention provides:

(a) A protein or polypeptide substance polyethyleneglycoloxy grouprepresented by the following general formula:

    R.sub.1 --(OCH.sub.2 CH.sub.2).sub.n --O--

(wherein, R₁ represents an optionally substituted cholesteryl group; andn represents a positive integer which is arbitrarily variable);

(b) A method for producing a protein or peptide substance having leastone amino group bonded to a polyethyleneglycoloxy group represented bythe following general formula:

    R.sub.1 --(OCH.sub.2 CH.sub.2).sub.n --O--

, comprising the step of causing a reaction of the protein or peptidesubstance with a reactive polyethyleneglycol derivative represented bythe following general formula:

    R.sub.1 --(OCH.sub.2 CH.sub.2).sub.n --O--R.sub.2

wherein, R₁ represents an optionally substituted cholesteryl group; R₂represents any of the following formulae (a), (b) and (c): ##STR1##(wherein, R₃ represents OH, alkoxy group, acyloxy group, a halogen atomor R₁ --(OCH₂ CH₂)_(n) --O--); and n represents a positive integer whichis arbitrarily variable!; and

(c) A polyethylenglycol derivative represented by the following generalformula:

    R.sub.1 --(OCH.sub.2 CH.sub.2).sub.n --O--R.sub.2

wherein, R₁ represents an optionally substituted cholesteryl group; R₂represents any of the following formulae (a), (b) and (c): ##STR2##(wherein, R₃ represents OH, alkoxy group, acyloxy group, a halogen atomor R₁ --(OCH₂ CH₂)_(n) --O--); and n represents a positive integer whichis arbitrarily variable)!.

More specifically, the present invention was derived from studies on anovel PEG derivative for improving in-vivo behaviors of protein,polyaminoacid and polypeptide substances having a physiological ormedical activity. The present invention was completed on the basis offindings that, when using the novel PEG derivative having a terminal ofthe cholesterol structure, an interaction with polymer components inblood is achieved, and this improves the in-vivo behaviors of a medicalsubstance. The present invention makes it possible to solve bondinghindrance between a medical substance and a receptor or the like, whichis a problem in conventional PEG modification of a protein or peptidemedical substance, and to develop a medical substance exhibiting a highpharmacological activity.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a graph illustrating concentrations in blood ofPEG-derivative-modified insulin and the result of an interactionin-vitro test;

FIG. 2 shows a graph illustrating concentrations in blood ofPEG-derivative-modified SOD and the result of an interaction in-vitrotest;

FIG. 3 shows a graph illustrating the result of an in-vivo test onbehaviors in vivo of PEG-derivative-modified insulin;

FIG. 4 shows a graph illustrating the result of an in-vivo test onbehaviors in vivo of PEG-derivative-modified SOD; and

FIG. 5 shows a graph illustrating the result of an in-vivo test onpharmacological effect of PEG-derivative-modified SOD.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The protein or peptide substance of the present invention is produced bybonding a reactive PEG derivative having a cholesterol structure asdescribed above at a terminal and a modifier comprising aphysiologically active substance.

Regarding the general formula, R₁ in the polyethyleneglycoloxy grouprepresents a cholesteryl group which may have an arbitrary substituentso far as it does not impair chemical modification action. Appropriatesubstituents include, for example, alkyl group, alkenyl group, hydroxylgroup and alkoxyl group.

R₂ bonded to this polyethyleneglycoloxy group may have any of thepartial structures of R₃ as described above, and the halogen atom in R₃means chlorine, bromine, iodine or the like. R₃ may be OH, alkoxy groupor acyloxy group. In the formula, n represents a positive integer whichis arbitrarily variable, and should be up to about 500, or preferably upto 200, or more preferably, within a range of from about 10 to 100.

Now, the method for producing the foregoing protein or polypeptidesubstance modified with the foregoing PEG derivative of the presentinvention is described. It suffices to cause a reaction between a ratioof the protein or polypeptide substance of 1 mol and a ratio of thereactive PEG derivative according to the following formula of 1 mol orover:

    R.sub.1 --(OCH.sub.2 CH.sub.2).sub.n --O--R.sub.2

There is no particular limitation on the reaction solvent so far as itdoes not participate in the reaction. Preferable solvents include, forexample, phosphoric acid buffer solution, boric acid buffer solution,Tris. acid buffer solution and acetic acid buffer solution. An organicsolvent such as acetonitrile, dimethylsulfoxide, and dimethylformamide,which do not participate in the reaction without deactivating a proteinor polypeptide substance may be added. Any of the foregoing reactive PEGderivative and the protein or polypeptide substance may be added firstto the reaction solvent, or may be added simultaneously.

The reaction may be caused without adding a reaction solvent.

The reaction temperature may be at any level so far as it does not causedenaturation of the protein or polypeptide substance, and shouldpreferably be within a range of from about 0° to 40° C.

A reaction time within a range of from about 0.5 to 72 hours issufficient, and a time within a range of from about 1 to 24 hours isusually sufficient to cause progress of the reaction.

After the reaction, the reaction product is purified by an ordinarymethod for purifying a protein or polypeptide substance such asdialysis, salting-out, ultrafiltration, ion-exchange chromatography orelectrophoresis, thereby obtaining a chemically modified protein orpolypeptide target substance. The extent of modification of amino groupcan be confirmed by an amino acid analysis carried out, for example,after acid decomposition.

The foregoing reactive PEG derivative can be manufactured with apolyethyleneglycol monocholesterol (hereinafter abbreviated as "C-PEGderivative") available by causing polyethyleneoxide to react withtertiary hydroxyl group present in cholestrine (also known ascholesterol) as the starting material.

A commercially available product (eg., one made by Nihon EmulsionCompany) or a synthesis product thereof may be used as C-PEG.

More particularly, a C-PEG derivative is first dissolved or suspended inan organic solvent such as acetonitrile, toluene, xylene, benzene,tetrahydrofuran, dioxane, dimethylsulfoxide or dimethylformamide, whichmay be any solvent so far as it does not participate in the reaction,and subsequently, any of the compounds expressed by the followinggeneral formulae (a), (b) and (c) is caused to react with the resultantsolution or suspension in an amount of 1 mol or over relative to 1 molof the C-PEG derivative: ##STR3##

where, X represents a halogen atom such as chlorine, bromine or iodine.!

An organic basic catalyst such as trimethylamine or triethylamine may beadded in an amount of 1 mol relative to 1 mol of the C-PEG derivative.

An inorganic base such as sodium hydroxide, potassium hydroxide oranhydrous sodium carbonate may be used: in this case, hydrogen in thehydroxide group of the C-PEG derivative is first substituted with sodiumin the foregoing solvent, together with the C-PEG derivative, and then,any of the compounds of the above-mentioned general formulae (a), (b)and (c) is added for reaction.

The reaction temperature should preferably be within a range of fromabout 0° to 300° C. or more preferably, from about 10° to 150° C.

The reaction temperature may be within a range of from several minutesto 24 hours.

The produced compound is purified by an ordinary chemical treatment suchas extraction, concentration, recrystallization, reprecipitation, columnchromatography, or distillation, thereby obtaining the foregoingreactive PEG derivative.

The water-soluble PEG derivative should preferably be selected frompolyethyleneglycol and homopolymers of polyethyleneglycol having ahydroxide group in the structure thereof, or more preferably, fromwater-soluble polymers such as polyethyleneglycol and derivativethereof.

The water-soluble PEG derivative used in the present invention shouldpreferably have a molecular weight within a range of from 100 to100,000, although there is no particular restrictions, or morepreferably, from 500 to 10,000.

In the present invention, the modified portion of the protein or peptidesubstance to be modified by the PEG derivative having a terminal of thecholesterol structure should preferably be an amino group, particularlya primary amino group.

Regarding the physiologically active substance modified by the PEGderivative having the cholesterol structure at the terminal thereof,there is no particular limitation imposed on the kind thereof in thepresent invention. Particularly for a protein or peptide medicalsubstance, preferable substances include, for example, substances P(hypothalamic hormone); corticotropin, lipotropin, melanotropin,vasopressin (neurohypophysial hormone), parathyroid hormone (thyroidhormone); thymopetine (thymus hormone); insulin, glucagon (pancreashormone); nerve growth factor, epidermal growth factor, insulin-likegrowth factor, human transforming/growth factor, growthhormone-releasing factor, i.e., CRF, fibroblast growth factor (growthfactor); secretin, cholecystokinin, vasoactive intestinal polypeptide,motilin (gastrointestinal hormone); gonadotropin (ciliary hormone);gonadotropic hormone-releasing hormone (gonadotropic hormone); relaxin(ovarian hormone); blood coagulation factor i-factor and u-factor(hemophilia factor); streptokinase, fibrinolysin, deoxyribonuclease,superoxide dismutase (hereinafter abbreviated as "SOD"), bilirubinoxidase, elastase, asparaginase (enzyme); tissue plasminogen activator,i.e., t-PA, urokinase (plasminogen); lymphokine (eg., interleukin),interferon, granulocyte colony-stimulating factor, i.e., G-CSF,macrophage colony-stimulating factor, i.e., M-CSF, granulocytemacrophage colony-stimulating factor, i.e., GM-CSF (stimulating factor);erythropoietin (hematopoietic factor), calcitonin, calcitoningene-related peptide (Ca-regulatory hormone); atrial natriuretic peptide(diuretic hormone); monoclonal and polyclonal antibodies; immunogen;enzyme-inhibiting factor; various polyamino acids includingpoly-L-lysine and poly-D-lysine; virus-derived cell membrane fusingpeptides; suhistone, protamine )gene binding protein) andanalog-structured substances having a physiological activity similar tothat of the foregoing protein or peptide drugs. These protein or peptidedrugs may be formed by macerating from a natural source or cellssubjected to a genetic engineering treatment, or through any of variousin-vitro synthetic methods. The terms in parentheses in theabove-enumerated examples show main uses of the protein or peptidemedical substances. It is needless to mention that the protein orpeptide substances used in the present invention include various aminoacids generically called polyaminoacids.

In the present invention, the number of modified points in thephysiologically active substance to be modified by the PEG derivativehaving the cholesterol structure at the terminal thereof, though notparticularly limited, should preferably be within a range of from 1 to100, or more preferably, from 1 to 10.

In the present invention, the protein or peptide substances are modifiedby the use of the reactive PEG derivative having the specific chemicalstructure as described above, whereby the in-vivo behaviors of thesemedical substances having a high physiological activity are effectivelyimproved without impairing bonding with a receptor or the like.

EXAMPLES

Now, the features and effects of the present invention will be describedfurther in detail by means of examples.

Example 1

Synthesis of a novel PEG derivative having the cholesterol structure atthe terminal thereof (polyethyleneglycol-monocholesterolether)!

(1)

After dissolving polyethyleneglycol-monocholesterolether (n=10, averagemolecular weight: 800, 9.5 g) and p-nitrophenyl chloroformate (0.6 g) intoluene (59 ml), triethylamine (0.3 g) was added, and the mixture wasstirred at the room temperature for 18 hours. Precipitated impuritieswere filtered off and the filtrate was concentrated under reducedpressure. The resultant residue was purified through a silica gelcolumn, thereby obtaining p-nitrophenyl formate-activatedpolyethylene-glycolmonocholesterolether (compound A) in an amount of 7.1g (yield: 75%).

Melting point: 23° C.

Elemental analysis: C₅₄ H₃₁ NO₁₅

                  TABLE 1    ______________________________________            C           H      N    ______________________________________    Calculated              65.23         9.23   1.41    Measured  65.15         9.46   1.38    ______________________________________

(2)

Polyethyleneglycol-monocholesterolether (n=20, average molecular weight:1,200, 6.0 g), succinic anhydride (0.6 g) and pyridine (0.5 ml) weredissolved in dichloromethane (30 ml) and circulated for three days.Subsequently, precipitated impurities were filtered off, and thefiltrate was evaporated. After dissolving the resultant residue indistilled water (50 ml), extraction was conducted with chloroform (50ml), and the resultant organic layer was evaporated, thereby obtainingpoly-ethyleneglycol-monocholesterolether succinate ester. Then, theresultant polyethyleneglycolmonocholesterolether succinate ester andN-succinimide (0.6 g) were dissolved in dimethylformamide (100 ml).After cooling the reaction system to 0° C., dichlorohexylcarbodiimide(0.7 g) dissolved in dimethylformamide (5 ml) was dripped and stirringwas continued at room temperature for 24 hours. The precipitatedimpurities were filtered off and the filtrate was concentrated under areduced pressure. Then, the resultant residue was purified through asilica gel column, thereby obtaining N-succinimide -activatedpolyethyleneglycol-monocholesterolether (compound B) in an amount of 4.8g (yield: 78%).

Melting point: 33° C.

Elemental analysis: C₇₅ H₁₃₅ NO₂₆

                  TABLE 2    ______________________________________            C           H      N    ______________________________________    Calculated              61.41         9.28   0.95    Measured  61.29         9.69   0.88    ______________________________________

(3)

After dissolving polyethyleneglycol-monocholesterolether (n=30, averagemolecular weight: 1,700, 4.5 g) and p-nitrophenylchloroformate (0.6 g)in toluene (50 ml), triethylamine (0.3 g) was added, and the mixture wasstirred at the room temperature for 18 hours. The precipitatedimpurities were filtered off and the filtrate was concentrated under areduced pressure. The resultant residue was purified through a silicagel column, thereby obtaining p-nitrophenyl formate-activatedpolyethylene-glycol-monocholesterolether (compound C) in an amount of3.8 g (yield: 81%).

Melting point: 36° C.

Elemental analysis: C₉₄ H₁₇₁ NO₃₉

                  TABLE 3    ______________________________________            C           H      N    ______________________________________    Calculated              58.21         8.89   0.72    Measured  58.12         8.95   0.68    ______________________________________

(4)

After dissolving anhydrous sodium carbonate (2.5 g) and cyanulchloride(1.3 g) in benzene anhydride (100 ml),polyethyleneglycol-monocholesterolether (n=30, average molecular weight:1,700, 4.5 g) was added, and the mixture was stirred at the roomtemperature for 24 hours. Then, triethylamine (0.3 g) was added, and themixture was stirred at the room temperature for 18 hours. Theprecipitated impurities were filtered off, and the filtrate wasconcentrated under a reduced pressure. The resultant residue waspurified through a silica gel column, thereby obtainingcyanulchloride-activated polyethylene-glycol-monocholesterolether(compound D) in an amount of 3.4 g (yield: 76%) .

Melting point: 38° C.

Elemental analysis: C₉₀ H₁₆₇ N₃ O₃₁ Cl₂

                  TABLE 4    ______________________________________            C           H      N    ______________________________________    Calculated              58.17         9.06   2.26    Measured  58.05         9.11   2.15    ______________________________________

(5)

After dissolving anhydrous sodium carbonate (2.5 g) and cyanulchloride(1.3 g) in benzene anhydride (100 ml),polyethyleneglycol-monocholesterolether (n=30, average molecular weight:1,700, 9.0 g) was added, and the mixture was stirred at the roomtemperature for 24 hours. Then, triethylamine (0.3 g) was added, and theresultant mixed solution was further stirred at the room temperature for18 hours. The precipitated impurities were filtered off, and thefiltrate was concentrated under a reduced pressure. The resultantresidue was purified through a silica gel column, thereby obtainingdouble-type cyanulchloride-activatedpolyethyleneglycol-monocholesterolether (compound E) in an amount of 5.8g (yield: 63%).

Melting point: 37° C.

Elemental analysis: C₁₇₇ H₃₃₄ N₃ O₆₂ Cl

                  TABLE 5    ______________________________________            C           H      N    ______________________________________    Calculated              60.18         9.53   1.18    Measured  60.06         9.64   1.07    ______________________________________

(6)

After dissolving polyethyleneglycol-monocholesterolether (n=100, averagemolecular weight: 4,700, 12.5 g) and p-nitrophenyl chloroformate (0.6 g)in toluene (50 ml), triethylamine (0.3 g) was added, and the mixture wasstirred at the room temperature for 18 hours. The precipitatedimpurities were filtered off, and the filtrate was concentrated under areduced pressure. The resultant residue was purified through a silicagel column, thereby obtaining p-nitrophenyl formate-activatedpoly-ethyleneglycol-monocholesterolether (compound F) in an amount of9.8 g (yield: 78%).

Melting point: 45° C.

Elemental analysis: C₂₃₄ H₄₅₁ NO₁₀₅

                  TABLE 6    ______________________________________            C           H      N    ______________________________________    Calculated              56.67         9.16   0.28    Measured  56.54         9.25   0.21    ______________________________________

Example 2

Modification of insulin with novel PEG derivative!

After dissolving bovine-derived insulin (6.0 mg) in 0.025 mM Na₂ B₄O₇.10H₂ O (pH: 9.2) in an amount of 200 ml, p-nitrophenylformate-activated polyethyleneglycolmono-cholesterolether (compound A),N-succinimide-activated polyethyleneglycol-monocholesterolether(compound B), p-nitrophenyl formate-activatedpolyethyleneglycol-mono-cholesterolether (compound C),cyanulchloride-activated polyethyleneglycol-monocholesterolether(compound D), double-type cyanulchloride-activatedpolyethyleneglycol-monocholesterolether (compound E), and p-nitrophenylformate-activated polyethyleneglycol-monocholosterolether (compound F)obtained in Example 1 were respectively added in an amount of 2.0 nmol,and the mixtures were stirred at the room temperature for five hours.The resultant mixtures were purified through gel filtration by the useof Sephadex G-75 (made by Pharmacia Company). The target fraction wasdesalted and concentrated through ultrafiltration (made by PharmaciaCompany), thereby obtaining the target aqueous solution (1.8 mg/ml). Inthe case of p-nitrophenyl formate-activatedpolyethylene-glycol-monocholesterolether (compoundA), the targetmodification was insufficient. This is attributable to the lowwater-solubility. In the case of p-nitrophenyl formate-activatedpolyethyleneglycol-monocholesterolether (compound F) also, the chemicalmodification was not sufficient. This is considered to be due tostereo-hindrance. In the other cases of PEG derivative-modified insulin,elution was observed in fractions corresponding to the molecular weightwhen a few points per molecule of insulin were modified.

Example 3

Modification of SOD with novel PEG derivative!

After dissolving bovine-derived Cu, Zn--SOD (Superoxide Dismutase) (30mg) in 0.025 mM Na₂ B₄ O₇.10H₂ O (pH: 9.2) in an amount of 30 ml, 3.9 mgp-nitrophenyl formate-activated polyethyleneglycol-monocholesterolether(compound C) obtained in Example 3 were added, and the mixture wasstirred at the room temperature for five hours. Then, the mixture waspurified through gel filtration using Sephadex G-75 (made by PharmaciaCompany). The target fraction was desalted and concentrated throughultrafiltration (made by Pharmacia Company), thereby obtaining a targetaqueous solution (20.2 mg/ml).

Example 4

In-vivo experiment on components in blood and interaction of novel PEGderivative-modified insulin!

Aqueous insulin solution, in an amount of 50 μl, modified withp-nitrophenyl-activated polyethyleneglycolmonocholesterolether (compoundC) obtained in Example 2 was added to 450 μl serum taken from a Wistermale rat or aqueous 4% rat albumin solution, and after stirring at 37°C. for 30 minutes, gel filtration-separation was conducted by the use ofSepharose 4B (made by Pharmacia Company). The result is shown in FIG. 1.An increase in the molecular weight of the novel PEG derivative-modifiedinsulin was confirmed only in the case of mixing with rat serum, and thepresence of interaction was confirmed with serum components other thanalbumin.

Example 5

In-vivo experiment on components in blood and interaction of novel PEGderivative-modified SOD!

Aqueous solution, in an amount of 50 μl, of SOD modified withp-nitrophenyl formate-activated poly-ethyleneglycolmonocholesterolether(compound C) obtained in Example 3 was added to 450 μl serum taken froma rat or 4% aqueous rat albumin solution, and after stirring at 37° C.for 30 minutes, the mixture was subjected to gel filtration-separationby the use of Sepharose 4B (made by Pharmacia Company). The result isshown in FIG. 2. An increase in the molecular weight of the novel PEGderivative-modified SOD was confirmed only in the case of mixing withrat serum, and the presence of interaction was confirmed with serumcomponents other than albumin.

Example 6

In-vivo experiment on in-vivo behaviors of insulin modified with novelPEG derivative!

About 0.5 U p-nitrophenyl formate-activatedpoly-ethyleneglycol-monocholesterolether (compound C)-modified ornon-modified insulin were intravenously administered to anurethane-anesthetized rat or a rat of which only the renal artery wasligated after urethane anesthesia. Then, at prescribed intervals oftime, venous blood was sampled to measure the insulin concentration inblood and blood glucose value at each time point. The result is shown inFIG. 3. The in-vivo behaviors of the modified and non-modified insulinin the normal rat were the same as the in-vivo behaviors of non-modifiedinsulin in the rat having the ligated renal artery. This result suggeststhat, for the modified insulin, the interaction with a clearancereceptor participating in disappearance from the biological body is nothindered by the modification. From the fact that, in the normal rat, themodified insulin and the non-modified insulin had identicalpharmacological effects, it is confirmed that, in the novel PEGderivative-modified insulin, the interaction with the insulin receptorassociated with the pharmacological functions participating indisappearance from the biological body is not impaired by themodification.

Example 7

In-vivo experiment on in-vivo behaviors of novel PEG derivative-modifiedSOD!

About 5,000 U p-nitrophenyl formate-activatedpolyethyleneglycol-monocholesterolether (compound C)-modified ornon-modified SOD were intravenously administered to anurethane-anesthetized rat or a rat of which only the renal artery wasligated after urethane anesthesia. Then, at prescribed intervals oftime, venous blood was sampled to measure the SOD concentration in bloodat each time point. The result is shown in FIG. 4. The result suggeststhat the in-vivo behaviors of the non-modified SOD in the normal rat areclearly improved, and glomerular filtration plays an important role indisappearance of non-modified SOD from the biological body. On the otherhand, it is confirmed that the in-vivo behaviors of the modified SOD arelargely improved: the half-life in blood is extended, and theinteraction with components in blood caused by modification allows themodified SOD to be exempt from glomerular filtration.

Example 8

In-vivo experiment on pharmacological effect of novel PEGderivative-modified SOD!

To a rat, 10,000 U p-nitrophenyl formate-activatedpolyethyleneglycol-monocholesterolether (compound C)-modified ornon-modified SOD were administered. Then, an acute gastric ulcer wascaused in accordance with the method for preparing a water immersionstress ulcerative model, and the effect inhibiting generation of ulcerat this point was investigated. The result is shown in FIG. 5. It isconfirmed from this result that, in the case of administration of themodified SOD, as compared with administration of the non-modified SOD,production of an ulcer is inhibited, and the modification intensifiesthe pharmacological effect in vivo.

INDUSTRIAL APPLICABILITY

By using the PEG derivative of the present invention, in-vivo behaviorsof physiologically active substance, particularly protein, polyaminoacidand polypeptide substances are improved, making it possible to developmedical substance exhibiting a high pharmacological effect, and at thesame time, it becomes possible to improve water-solubility of thesedrugs, increase storage stability thereof, reduce immunogenicity, andimprove resistance to enzymatic degradation.

The PEG derivative-modified protein or polypeptide substances of thepresent invention can be administered to mammals (such as dogs, pigs,cows, horses and persons), through oral or parenteral administration, inthe form of tablets, capsules, injection and other medical compositionsby using a known carrier or diluent.

Or, for example, the PEG derivative-modified insulin of the presentinvention has a function of reducing blood glucose, and can therefore beadministered as an injection in an amount within a range of from 4 to100 units per day as a treatment drug or a preventive agent againstdiabetes mellitus.

The peg derivative-modified sod, having an antiulcer effect, isapplicable as an antiulcer agent against stomach or duodenum, or havingan anti-inflammatory effect, is applicable as an anti-inflammatoryagent, and can be administered in an amount of from 1 to 5 mg/kg per dayin the form of tablets or injection.

What is claimed is:
 1. A modified polypeptide comprising a polypeptidehaving an amino group bonded to a polyethyleneglycoloxy grouprepresented by the following general formula:

    R.sub.1 --(OCH.sub.2 --CH.sub.2).sub.n --O--

wherein R₁ represents a cholesteryl group, and n represents a positiveinteger up to
 500. 2. The modified polypeptide according to claim 1,wherein the polypeptide is insulin.
 3. The modified polypeptideaccording to claim 1, wherein the polypeptide is superoxide dismutase.4. A method for producing the modified polypeptide of claim 1,comprising the step of reacting the polypeptide with a reactivepolyethyleneglycol represented by the following general formula:

    R.sub.1 --(OCH.sub.2 --CH.sub.2).sub.n --O--R.sub.2

wherein R₁ represents a cholesteryl group, n represents a positiveinteger up to 500, and R₂ represents any of the following formulae (a),(b) and (c): ##STR4## wherein R₃ represents OH, alkoxy group, acyloxygroup, a halogen atom or R₁ --(OCH₂ CH₂)_(n) --O--, wherein R₁ and nhave the same meanings as defined above.
 5. The method of claim 4,wherein the polypeptide is insulin or superoxide dismutase.
 6. Areactive polyethyleneglycol derivative represented by the followinggeneral formula:

    R.sub.1 --(OCH.sub.2 --CH.sub.2).sub.n --O--R.sub.2

wherein R₁ represents a cholesteryl group, n represents a positiveinteger up to 500, and R₂ represents any of the following formulae (a),(b) and (c): ##STR5## wherein R₃ represents OH, alkoxy group, acyloxygroup, a halogen atom or R₁ --(OCH₂ CH₂)_(n) --O--, wherein R₁ and nhave the same meanings as defined above.
 7. A composition for thetreatment of diabetes mellitus comprising the modified polypeptide ofclaim 2 and a pharmaceutically acceptable carrier or diluent.
 8. Acomposition for the treatment of ulcer comprising the modifiedpolypeptide of claim 3 and a pharmaceutically acceptable carrier ordiluent.
 9. The modified polypeptide according to claim 1, wherein R₁represents a cholesteryl group which is substituted with a substituentselected from the group consisting of alkyl, alkenyl, hydroxyl andalkoxyl.
 10. The method according to claim 4, wherein R₁ represents acholesteryl group which is substituted with a substituent selected fromthe group consisting of alkyl, alkenyl, hydroxyl and alkoxyl.
 11. Thereactive derivative according to claim 6, wherein R₁ represents acholesteryl group which is substituted with a substituent selected fromthe group consisting of alkyl, alkenyl, hydroxyl and alkoxyl.
 12. Apharmaceutical composition comprising the modified polypeptide accordingto claim 1 and a pharmaceutically acceptable carrier or diluent.